The canonical chemical synapse of the nervous system is characterized by electron-dense thickening of the post-synaptic membrane in close apposition to the pre-synaptic active zone. Quite distinct and far less well- understood are the so-called c-synapses named for the sub-synaptic cistern in the post-synaptic cell that is aligned with the pre-synaptic terminal. Although common on motor neurons and cerebellar Purkinje cells among others, essentially nothing is known about c-synapse function except that they are formed by cholinergic inputs. Chief among the mysteries is the role of the subsynaptic cistern itself, although its similarity to sarcoplasmic reticulum has prompted suggestions of involvement in calcium (Ca2+) signaling. The goal of this proposal is to advance understanding of c-synapse function through a combination of computational modeling coupled with experiments on the cholinergic c-synapse. A major impediment to study of c-synapses on central neurons is the fact that there is typically no means to activate them selectively out of th multitude of inputs. We will take advantage of an exemplar c-synapse that is experimentally tractable - efferent cholinergic neurons that project from the brainstem medial olivocochlear nucleus to inhibit cochlear outer hair cells (OHCs). Because this is its sole synaptic input, the OHC will provide unique insights into how c-synapses operate. First and foremost is to determine whether the cistern regulates postsynaptic Ca2+. Remarkably, c- synapses share common design features with the fundamental structural units of excitation-contraction coupling in the heart known as dyads. In each case, a Ca2+ store (junctional sarcoplasmic reticulum JSR in heart; cistern in the OHC) is positioned close (~14 nm) to the cell membrane, creating a restricted Ca2+ nano- domain. Ca2+ sources in the cell membrane (voltage-gated Ca2+ channels in heart; nicotinic cholinergic receptors nAChRs in OHCs) direct their flux into this restricted space. In OHCs, Ca2+-activated potassium type- 2 (SK2) channels are located near the nAChRs. Even small fluxes directed into the cleft can create large Ca2+ signals that are highly localized in space and time. Ryanodine-sensitive, Ca2+-binding Ca2+-release channels (RyRs) are thought to reside in the closely apposed cistern membrane as in JSR, suggesting that the process of Ca2+-induced Ca2+-release (CICR) may be important to OHC function, as it is in the heart. We will leverage these similarities to harness extensive modeling work done in the Winslow lab on CICR in the heart, adapting these models and applying them to advance our understanding of the function of c-synapses in the nervous system. Modeling work will be informed by experiments conducted in the Fuchs lab that will probe the structure and function of the OHC c-synapses. This unique combination of an experimentally-tractable system, along with the modeling and experimental expertise of these two labs, will enable us to advance our understanding of the function of c-synapses in the nervous system.